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CRISPR/Cas9-mediated endogenous protein tagging for RESOLFT super-resolution microscopy of living human cells.

Ratz M, Testa I, Hell SW, Jakobs S - Sci Rep (2015)

Bottom Line: We demonstrate the benefit of endogenous expression levels compared to overexpression and show that typical overexpression-induced artefacts were avoided in genome-edited cells.Fluorescence activated cell sorting analysis revealed a narrow distribution of fusion protein expression levels in genome-edited cells, compared to a pronounced variability in transiently transfected cells.Our strategy to generate human cell lines expressing fluorescent fusion proteins at endogenous levels for RESOLFT nanoscopy can be extended to other fluorescent tags and super-resolution approaches.

View Article: PubMed Central - PubMed

Affiliation: Max Planck Institute for Biophysical Chemistry, Department of NanoBiophotonics, Am Fassberg 11, 37077 Göttingen, Germany.

ABSTRACT
Overexpression is a notorious concern in conventional and especially in super-resolution fluorescence light microscopy studies because it may cause numerous artifacts including ectopic sub-cellular localizations, erroneous formation of protein complexes, and others. Nonetheless, current live cell super-resolution microscopy studies generally rely on the overexpression of a host protein fused to a fluorescent protein. Here, we establish CRISPR/Cas9-mediated generation of heterozygous and homozygous human knockin cell lines expressing fluorescently tagged proteins from their respective native genomic loci at close to endogenous levels. We tagged three different proteins, exhibiting various localizations and expression levels, with the reversibly switchable fluorescent protein rsEGFP2. We demonstrate the benefit of endogenous expression levels compared to overexpression and show that typical overexpression-induced artefacts were avoided in genome-edited cells. Fluorescence activated cell sorting analysis revealed a narrow distribution of fusion protein expression levels in genome-edited cells, compared to a pronounced variability in transiently transfected cells. Using low light intensity RESOLFT (reversible saturable optical fluorescence transitions) nanoscopy we show sub-diffraction resolution imaging of living human knockin cells. Our strategy to generate human cell lines expressing fluorescent fusion proteins at endogenous levels for RESOLFT nanoscopy can be extended to other fluorescent tags and super-resolution approaches.

No MeSH data available.


Related in: MedlinePlus

CRISPR/Cas9-mediated knockin of rsEGFP2 at three genomic loci in human U2OS cells.(a) Workflow for the generation of monoclonal human knockin cell lines for RESOLFT super-resolution microscopy. (b) Schematic representation of the integration strategy for generating C-terminally tagged rsEGFP2 fusion proteins expressed from the endogenous locus. White boxes, 5′- and 3′-untranslated region (UTR); blue boxes, exons; ATG, start codon; TAA, stop codon; HDR, homology-directed repair; F, locus-specific forward primer; R, locus-specific reverse primer; GR, rsEGFP2-specific reverse primer. (c, d) Analysis of two clonal lines per target locus. (c) Out-out PCR using primers F and R probing for locus-specific integration. CTL, control (parental U2OS cells); HMGA1-HET, heterozygous HMGA1-rsEGFP2HET1.5 clone, HMGA1-HOM, homozygous HMGA1-rsEGFP2HOM2.4 clone; ZYX-HET, heterozygous ZYX-rsEGFP2HET clone; ZYX-HOM, homozygous ZYX-rsEGFP2HOM clone; VIM-HET1, heterozygous VIM-rsEGFP2HET1.2 clone; VIM-HET2, heterozygous VIM-rsEGFP2HET2.1 clone. (d) Junction PCR using primers F and GR probing for locus-specific integration of rsEGFP2 transgene. (e–g) Western blot analysis of cell lysates of monoclonal cell lines immunoblotted for rsEGFP2, beta-Actin and the respective endogenously tagged protein: HMG-I (e), Zyxin (f) and Vimentin (g). Full length blots are shown in Supplementary Fig. 10.
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f1: CRISPR/Cas9-mediated knockin of rsEGFP2 at three genomic loci in human U2OS cells.(a) Workflow for the generation of monoclonal human knockin cell lines for RESOLFT super-resolution microscopy. (b) Schematic representation of the integration strategy for generating C-terminally tagged rsEGFP2 fusion proteins expressed from the endogenous locus. White boxes, 5′- and 3′-untranslated region (UTR); blue boxes, exons; ATG, start codon; TAA, stop codon; HDR, homology-directed repair; F, locus-specific forward primer; R, locus-specific reverse primer; GR, rsEGFP2-specific reverse primer. (c, d) Analysis of two clonal lines per target locus. (c) Out-out PCR using primers F and R probing for locus-specific integration. CTL, control (parental U2OS cells); HMGA1-HET, heterozygous HMGA1-rsEGFP2HET1.5 clone, HMGA1-HOM, homozygous HMGA1-rsEGFP2HOM2.4 clone; ZYX-HET, heterozygous ZYX-rsEGFP2HET clone; ZYX-HOM, homozygous ZYX-rsEGFP2HOM clone; VIM-HET1, heterozygous VIM-rsEGFP2HET1.2 clone; VIM-HET2, heterozygous VIM-rsEGFP2HET2.1 clone. (d) Junction PCR using primers F and GR probing for locus-specific integration of rsEGFP2 transgene. (e–g) Western blot analysis of cell lysates of monoclonal cell lines immunoblotted for rsEGFP2, beta-Actin and the respective endogenously tagged protein: HMG-I (e), Zyxin (f) and Vimentin (g). Full length blots are shown in Supplementary Fig. 10.

Mentions: We established a workflow (Fig. 1a) to create cell lines stably expressing C-terminal rsEGFP2 fusion proteins from the respective endogenous promotors using the CRISPR/Cas9 system (Fig. 1b). Specifically, we tagged the nuclear DNA-binding non-histone high mobility group protein HMG-I (gene: HMGA1), the class-III intermediate filament protein Vimentin (gene: VIM), and the focal adhesions plaque protein Zyxin (gene: ZYX). We choose these proteins for this study because they exhibit different expression levels and are localized in different cellular compartments, i.e. the nucleus (HMG-I), the cytoskeleton (Vimentin) and in the plasma membrane associated focal adhesion complexes (Zyxin). For each of the three target genes we designed two gRNAs (Supplementary Table 1) and one donor matrix (Supplementary Table 2) bearing homology arms of about 600 to 900 bp length to facilitate the integration of the rsEGFP2 coding sequence at the 3′-end of the respective last exon, replacing the stop codon but leaving the genomic locus otherwise unchanged. After co-transfection of human U2OS cells with a bicistronic plasmid encoding the gRNA and Cas9 together with the corresponding donor plasmid, single rsEGFP2-positive cells were sorted by fluorescence activated cell sorting (FACS) into 96-well plates. Of the six nuclease/donor matrix pairs tested, only gRNA2 targeting ZYX failed to result in cells expressing the expected fusion protein as judged by fluorescence microscopy prior to FACS. For all other gRNAs used, FACS-analysis revealed that between 0.1% and 4.7% of the transfected U2OS cells displayed a clearly discernible rsEGFP2 fluorescence signal (Supplementary Fig. 1). On average, between 10% and 20% of the sorted rsEGFP2 expressing single cells recovered and grew to confluency in the 96-well plate. This efficiency presumably could be further increased by supplementing the growth medium with antioxidants such as α-thioglycerol or bathocuprione disulphonate1920.


CRISPR/Cas9-mediated endogenous protein tagging for RESOLFT super-resolution microscopy of living human cells.

Ratz M, Testa I, Hell SW, Jakobs S - Sci Rep (2015)

CRISPR/Cas9-mediated knockin of rsEGFP2 at three genomic loci in human U2OS cells.(a) Workflow for the generation of monoclonal human knockin cell lines for RESOLFT super-resolution microscopy. (b) Schematic representation of the integration strategy for generating C-terminally tagged rsEGFP2 fusion proteins expressed from the endogenous locus. White boxes, 5′- and 3′-untranslated region (UTR); blue boxes, exons; ATG, start codon; TAA, stop codon; HDR, homology-directed repair; F, locus-specific forward primer; R, locus-specific reverse primer; GR, rsEGFP2-specific reverse primer. (c, d) Analysis of two clonal lines per target locus. (c) Out-out PCR using primers F and R probing for locus-specific integration. CTL, control (parental U2OS cells); HMGA1-HET, heterozygous HMGA1-rsEGFP2HET1.5 clone, HMGA1-HOM, homozygous HMGA1-rsEGFP2HOM2.4 clone; ZYX-HET, heterozygous ZYX-rsEGFP2HET clone; ZYX-HOM, homozygous ZYX-rsEGFP2HOM clone; VIM-HET1, heterozygous VIM-rsEGFP2HET1.2 clone; VIM-HET2, heterozygous VIM-rsEGFP2HET2.1 clone. (d) Junction PCR using primers F and GR probing for locus-specific integration of rsEGFP2 transgene. (e–g) Western blot analysis of cell lysates of monoclonal cell lines immunoblotted for rsEGFP2, beta-Actin and the respective endogenously tagged protein: HMG-I (e), Zyxin (f) and Vimentin (g). Full length blots are shown in Supplementary Fig. 10.
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Related In: Results  -  Collection

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f1: CRISPR/Cas9-mediated knockin of rsEGFP2 at three genomic loci in human U2OS cells.(a) Workflow for the generation of monoclonal human knockin cell lines for RESOLFT super-resolution microscopy. (b) Schematic representation of the integration strategy for generating C-terminally tagged rsEGFP2 fusion proteins expressed from the endogenous locus. White boxes, 5′- and 3′-untranslated region (UTR); blue boxes, exons; ATG, start codon; TAA, stop codon; HDR, homology-directed repair; F, locus-specific forward primer; R, locus-specific reverse primer; GR, rsEGFP2-specific reverse primer. (c, d) Analysis of two clonal lines per target locus. (c) Out-out PCR using primers F and R probing for locus-specific integration. CTL, control (parental U2OS cells); HMGA1-HET, heterozygous HMGA1-rsEGFP2HET1.5 clone, HMGA1-HOM, homozygous HMGA1-rsEGFP2HOM2.4 clone; ZYX-HET, heterozygous ZYX-rsEGFP2HET clone; ZYX-HOM, homozygous ZYX-rsEGFP2HOM clone; VIM-HET1, heterozygous VIM-rsEGFP2HET1.2 clone; VIM-HET2, heterozygous VIM-rsEGFP2HET2.1 clone. (d) Junction PCR using primers F and GR probing for locus-specific integration of rsEGFP2 transgene. (e–g) Western blot analysis of cell lysates of monoclonal cell lines immunoblotted for rsEGFP2, beta-Actin and the respective endogenously tagged protein: HMG-I (e), Zyxin (f) and Vimentin (g). Full length blots are shown in Supplementary Fig. 10.
Mentions: We established a workflow (Fig. 1a) to create cell lines stably expressing C-terminal rsEGFP2 fusion proteins from the respective endogenous promotors using the CRISPR/Cas9 system (Fig. 1b). Specifically, we tagged the nuclear DNA-binding non-histone high mobility group protein HMG-I (gene: HMGA1), the class-III intermediate filament protein Vimentin (gene: VIM), and the focal adhesions plaque protein Zyxin (gene: ZYX). We choose these proteins for this study because they exhibit different expression levels and are localized in different cellular compartments, i.e. the nucleus (HMG-I), the cytoskeleton (Vimentin) and in the plasma membrane associated focal adhesion complexes (Zyxin). For each of the three target genes we designed two gRNAs (Supplementary Table 1) and one donor matrix (Supplementary Table 2) bearing homology arms of about 600 to 900 bp length to facilitate the integration of the rsEGFP2 coding sequence at the 3′-end of the respective last exon, replacing the stop codon but leaving the genomic locus otherwise unchanged. After co-transfection of human U2OS cells with a bicistronic plasmid encoding the gRNA and Cas9 together with the corresponding donor plasmid, single rsEGFP2-positive cells were sorted by fluorescence activated cell sorting (FACS) into 96-well plates. Of the six nuclease/donor matrix pairs tested, only gRNA2 targeting ZYX failed to result in cells expressing the expected fusion protein as judged by fluorescence microscopy prior to FACS. For all other gRNAs used, FACS-analysis revealed that between 0.1% and 4.7% of the transfected U2OS cells displayed a clearly discernible rsEGFP2 fluorescence signal (Supplementary Fig. 1). On average, between 10% and 20% of the sorted rsEGFP2 expressing single cells recovered and grew to confluency in the 96-well plate. This efficiency presumably could be further increased by supplementing the growth medium with antioxidants such as α-thioglycerol or bathocuprione disulphonate1920.

Bottom Line: We demonstrate the benefit of endogenous expression levels compared to overexpression and show that typical overexpression-induced artefacts were avoided in genome-edited cells.Fluorescence activated cell sorting analysis revealed a narrow distribution of fusion protein expression levels in genome-edited cells, compared to a pronounced variability in transiently transfected cells.Our strategy to generate human cell lines expressing fluorescent fusion proteins at endogenous levels for RESOLFT nanoscopy can be extended to other fluorescent tags and super-resolution approaches.

View Article: PubMed Central - PubMed

Affiliation: Max Planck Institute for Biophysical Chemistry, Department of NanoBiophotonics, Am Fassberg 11, 37077 Göttingen, Germany.

ABSTRACT
Overexpression is a notorious concern in conventional and especially in super-resolution fluorescence light microscopy studies because it may cause numerous artifacts including ectopic sub-cellular localizations, erroneous formation of protein complexes, and others. Nonetheless, current live cell super-resolution microscopy studies generally rely on the overexpression of a host protein fused to a fluorescent protein. Here, we establish CRISPR/Cas9-mediated generation of heterozygous and homozygous human knockin cell lines expressing fluorescently tagged proteins from their respective native genomic loci at close to endogenous levels. We tagged three different proteins, exhibiting various localizations and expression levels, with the reversibly switchable fluorescent protein rsEGFP2. We demonstrate the benefit of endogenous expression levels compared to overexpression and show that typical overexpression-induced artefacts were avoided in genome-edited cells. Fluorescence activated cell sorting analysis revealed a narrow distribution of fusion protein expression levels in genome-edited cells, compared to a pronounced variability in transiently transfected cells. Using low light intensity RESOLFT (reversible saturable optical fluorescence transitions) nanoscopy we show sub-diffraction resolution imaging of living human knockin cells. Our strategy to generate human cell lines expressing fluorescent fusion proteins at endogenous levels for RESOLFT nanoscopy can be extended to other fluorescent tags and super-resolution approaches.

No MeSH data available.


Related in: MedlinePlus